Basic Study
Copyright ©The Author(s) 2020.
World J Clin Infect Dis. May 15, 2020; 10(1): 14-23
Published online May 15, 2020. doi: 10.5495/wjcid.v10.i1.14
Table 1 Bacterial strains and plasmids used in the study
Strain/plasmidDescriptionSource
Strain
DH5a MCRTransformation efficient strainGibco/BRL
MC4100E. coli K-12 strainLinda Kenney
EK227E. coli K-12 strain[53]
EF1007gadE::Km in EK227[54]
EF1083gadE::Km/pPCRScript Amp gadE[33]
NU149Clinical isolate[36]
NU149 gadEΔgadE mutation in NU149This study
NU149 LacZ1ΔlacZ mutation in NU149This study
Plasmid
pUJ9Promoterless lacZ gene, ApR[37]
pPP2-6Single copy plasmid, CmR[38]
pKD4Flp recombinase sites, KmR[40]
pKD46Red recombinase, ApR[40]
pCP20Flp recombinase, ApR[40]
pNK2-29fliC::lacZ on pPP2-6, ApRThis study
pPCRScript Amp gadEgadE on pPCRScript Amp[33]
Table 2 Effect of pH on fliC::lacZ gene transcription in Escherichia coli strain MC4100/pNK2-29 grown in buffered Luria Bertani media
pHGal activity1
5.5288 ± 81.5
6528 ± 82.5
6.5629 ± 114
71111 ± 110
7.5932 ± 190
8738 ± 125
Table 3 Effect of osmolarity on fliC::lacZ gene transcription in Escherichia coli grown in buffered pH 5.5 and 7.0 Luria Bertani media with different osmolarities
E. coli strainNaCl (mmol/L)Gal activity1
pH 5.5pH 7.0
MC4100/pNK2-290308 ± 10421132 ± 130
MC4100/pNK2-29100338 ± 128806 ± 41
MC4100/pNK2-29200251 ± 68.5689 ± 173
MC4100/pNK2-2940062 ± 22.0454 ± 71
NU149 LacZ1/pNK2-290442 ± 721353 ± 98
NU149 LacZ1/pNK2-29100418 ± 61976 ± 52
NU149 LacZ1/pNK2-29200293 ± 43811 ± 75
NU149 LacZ1/pNK2-29400147 ± 39489 ± 61
Table 4 Assessing a gadE and mutation and complementation on fliC::lacZ gene transcription in Escherichia coli grown in buffered pH 5.5 and 7.0 Luria Bertani media with different osmolarities
E. coli strainGal activity1
pH 5.5pH 5.52pH 7.0pH 7.0
EK227/pNK2-29540 ± 513165 ± 591520 ± 144540 ± 66
EF1007/pNK2-2941742 ± 109470 ± 1062196 ± 173681 ± 135
EF1083/pNK2-29295 ± 93131 ± 20794 ± 145404 ± 41
Table 5 Motility of Escherichia coli strains NU149 and EK227, their gadE mutants, and complemented gadE mutants grown in pH 5.5 Luria Bertani
StrainMotility (mm)1
NU14910.67 ± 1.252
NU149 gadE57.34 ± 10.21
NU149 gadE/pPCRScript gadE7.00 ± 0.82
EK2278.33 ± 1.52
EF1007 (gadE)45.00 ± 2.00
EF1083 (gadE/pPCRScript gadE)6.67 ± 1.53